Gene Expression System in Green Sulfur Bacteria by Conjugative Plasmid Transfer

<div><p>Gene transfer and expression systems in green sulfur bacteria were established by bacterial conjugation with <i>Escherichia coli</i>. Conjugative plasmid transfer from <i>E. coli</i> S17-1 to a thermophilic green sulfur bacterium, <i>Chlorobaculum tepidum</i> (formerly <i>Chlorobium tepidum</i>) WT2321, was executed with RSF1010-derivative broad-host-range plasmids, named pDSK5191 and pDSK5192, that confer erythromycin and streptomycin/spectinomycin resistance, respectively. The transconjugants harboring these plasmids were reproducibly obtained at a frequency of approximately 10^-5 by selection with erythromycin and a combination of streptomycin and spectinomycin, respectively. These plasmids were stably maintained in <i>C. tepidum</i> cells in the presence of these antibiotics. The plasmid transfer to another mesophilic green sulfur bacterium, <i>C. limnaeum</i> (formerly <i>Chlorobium phaeobacteroides</i>) RK-j-1, was also achieved with pDSK5192. The expression plasmid based on pDSK5191 was constructed by incorporating the upstream and downstream regions of the <i>pscAB</i> gene cluster on the <i>C. tepidum</i> genome, since these regions were considered to include a constitutive promoter and a ρ-independent terminator, respectively. Growth defections of the ∆<i>cycA</i> and ∆<i>soxB</i> mutants were completely rescued after introduction of pDSK5191-<i>cycA</i> and -<i>soxB</i> that were designed to express their complementary genes. On the other hand, pDSK5191-<i>6xhis-pscAB</i>, which incorporated the gene cluster of <i>6xhis-pscA</i> and <i>pscB</i>, produced approximately four times more of the photosynthetic reaction center complex with His-tagged PscA as compared with that expressed in the genome by the conventional natural transformation method. This expression system, based on conjugative plasmid, would be applicable to general molecular biological studies of green sulfur bacteria.</p> </div

Restriction enzyme mappings of the plasmids in the <i>C. tepidum</i> and <i>C. limnaeum</i> transconjugants.

<p>(A, B) Physical maps of the plasmids from the <i>C. tepidum</i> transconjugants. The maps were constructed with restriction enzymes <i>Eco</i>RI (A) and <i>Bgl</i>II (B). The restriction fragments were separated by agarose gel (1%) electrophoresis. The control plasmids pDSK5191 (lane 1), pDSK5191-<i>cycA</i> (lane 2), and pDSK5191-<i>soxB</i> (lane 3) were obtained from the donor S17-1 cultures. Lanes 4-11 are the plasmid samples of the <i>C. tepidum</i> cultures. The genotype of <i>C. tepidum</i> is indicated above each lane. The bars and numbers at the left side of the panel indicate mobility and size of the <i>Sty</i>I digests of the λ-phage DNA. (C) Physical maps of the plasmids from <i>C. limnaeum</i> transconjugants. The maps were constructed with restriction enzymes <i>Eco</i>RI (lanes 1–3) and <i>Bam</i>HI (lanes 4–6). Lanes 1 and 4 are pDSK5192 plasmids prepared from the donor S17-1 cultures. Lanes 2-3 and 5-6 are the plasmid samples of the <i>C. limnaeum</i> cultures. The genotype of <i>C. limnaeum</i> is indicated above each lane. The bars and numbers at the left side of the panel indicate mobility and sizes of the <i>Sty</i>I digests of the λ-phage DNA, respectively. </p

Schematic genetic maps of pDSK5191 and its derivative expression plasmids.

<p>(A) Genetic map of the IncQ-group conjugation plasmid pDSK5191. Protein coding sequences are shown as block arrows. The pale gray rectangle represents the region of Ω-cassette in which T4-phage transcription and translation terminator sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082345#B23" target="_blank">23</a>] are located at both ends. “Ori” represents the region containing the <i>oriV</i> and <i>oriT</i> sequences, which are derived from RSF1010 and are required for replication and mobilization of the plasmid, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082345#B15" target="_blank">15</a>]. (B) Expression constructs of the pDSK5191-derivative plasmids. Each construct was inserted into the unique <i>Eco</i>RI site of pDSK5191, as indicated by the arch-shaped arrow at the top of the panels. Protein-coding sequences are shown as block arrows. The pale gray rectangle represents the six-consecutive histidine-tag (6xhis) attached to the 5’ end of the <i>pscA</i> gene. “P_<i>pscA</i>” and “ter” are a putative constitutive promoter and a ρ-independent transcription terminator, respectively, of the <i>C. tepidum </i><i>pscAB</i> gene cluster.</p

Growth curves of <i>C. tepidum</i> mutants after gene complementation experiments.

<p>Growth curves of <i>C. tepidum</i> mutants used as host strains (A), transconjugant strains of ∆<i>cycA</i> mutant (B), and ∆<i>soxB</i> mutant (C). Each strain was grown in a liquid CL medium at 40°C (for details, see Materials and Methods), and its optical density (O.D.) was monitored at 660 nm. In the transconjugant cultures, 1 µg/mL of Em was added for the stable maintenance of plasmids. The average values and standard deviations, which were obtained from at least three independent experiments, were plotted.</p

Distal Regulation of Heme Binding of Heme Oxygenase‑1 Mediated by Conformational Fluctuations

Heme oxygenase-1 (HO-1) is an enzyme that catalyzes the oxidative degradation of heme. Since free heme is toxic to cells, rapid degradation of heme is important for maintaining cellular health. There have been useful mechanistic studies of the HO reaction based on crystal structures; however, how HO-1 recognizes heme is not completely understood because the crystal structure of heme-free rat HO-1 lacks electron densities for A-helix that ligates heme. In this study, we characterized conformational dynamics of HO-1 using NMR to elucidate the mechanism by which HO-1 recognizes heme. NMR relaxation experiments showed that the heme-binding site in heme-free HO-1 fluctuates in concert with a surface-exposed loop and transiently forms a partially unfolded structure. Because the fluctuating loop is located over 17 Å distal from the heme-binding site and its conformation is nearly identical among different crystal structures including catalytic intermediate states, the function of the loop has been unexamined. In the course of elucidating its function, we found interesting mutations in this loop that altered activity but caused little change to the conformation. The Phe79Ala mutation in the loop changed the conformational dynamics of the heme-binding site. Furthermore, the heme binding kinetics of the mutant was slower than that of the wild type. Hence, we concluded that the distal loop is involved in the regulation of the conformational change for heme binding through the conformational fluctuations. Similar to other enzymes, HO-1 effectively promotes its function using the identified distal sites, which might be potential targets for protein engineering

Specific Gene <i>bciD</i> for C7-Methyl Oxidation in Bacteriochlorophyll <i>e</i> Biosynthesis of Brown-Colored Green Sulfur Bacteria

<div><p>The gene named <i>bciD</i>, which encodes the enzyme involved in C7-formylation in bacteriochlorophyll <i>e</i> biosynthesis, was found and investigated by insertional inactivation in the brown-colored green sulfur bacterium <i>Chlorobaculum limnaeum</i> (previously called <i>Chlorobium phaeobacteroides</i>). The <i>bciD</i> mutant cells were green in color, and accumulated bacteriochlorophyll <i>c</i> homologs bearing the 7-methyl group, compared to C7-formylated BChl <i>e</i> homologs in the wild type. BChl-<i>c</i> homolog compositions in the mutant were further different from those in <i>Chlorobaculum tepidum</i> which originally produced BChl <i>c</i>: (3¹<i>S</i>)-8-isobutyl-12-ethyl-BChl <i>c</i> was unusually predominant.</p> </div

Construction of <i>Cba. limnaeum bciD</i> gene inactivated mutant.

<p>(A) Schematic map of genes arrangement around <i>bciD</i> gene in the genome of <i>Cba. limnaeum</i> RK-j-1, and its insertional inactivation. The <i>aadA1</i> gene, conferring resistance to streptomycin and spectinomycin, was inserted in <i>bciD</i>. Arrows represent the primers bciD-F (i), bciD-R (ii), bciD-inf-F (iii), bciD-inf-R (iv), bciD-comf-F (v), and bciD-comf-R (vi). (B) PCR confirmation of gene interruption. The <i>bciD</i> gene was amplified from genomic DNA extracted from the wild type (lanes 1 and 2) and a mutant (lanes 3 and 4) of <i>Cba. limnaeum</i>, using above bciD-comf-F and -R primers. The products in lanes 2 and 4 were then digested by restriction enzyme <i>Eco</i>RV, and the fragments yielded from wild type and the mutant were 1.36 and 0.78, and 2.22 kbp, respectively. Lane M, molecular size marker (the sizes of bands are indicated at left).</p

Proposed oxidization of a methyl group at the C7 position of BChlide <i>c</i> to a formyl group in BChl <i>e</i> biosynthesis:

<p>(i) monooxygenation of the 7-methyl to 7-hydroxymethyl group; (ii) monooxygenation of the 7-hydroxymethyl to 7-dihydroxymethyl group followed by the spontaneous dehydration to the 7-formyl group or direct dehydrogenation of the 7-hydroxymethyl to 7-formyl group. The C3¹-stereochemistry is <i>R</i> or <i>S</i>; R¹ =  ethyl, propyl, or isobutyl; R² =  methyl or ethyl.</p

UV-Vis-NIR absorption spectra of whole cells (A) and extracted pigments (B) of the <i>Cba. limnaeum</i> wild type (broken lines) and <i>bciD</i> mutant (solid lines); measured using a Hitachi UV-2550 (Shimadzu, Japan) spectrophotometer.

<p>(A) Wild type and mutant cells in stationary phase were collected and suspended in 50 mM Tris-HCl (pH 7.8) containing 150 mM NaCl; normalized at 660 nm. (B) Pigments were extracted with a mixture of acetone and methanol (7∶2, v/v), and used for the measurements; normalized at Soret maxima. The dotted line shows absorption spectrum of pigments from <i>Cba. tepidum</i> as control for BChl <i>c</i>.</p

HPLC elution profiles of extracted pigments from GSB.

<p>(A) From <i>Cba. tepidum</i> as control for BChl <i>c</i>, recorded at 435 nm. (B) From <i>Cba. limnaeum bciD</i> mutant recorded at 435 nm. (C) From <i>Cba. limnaeum</i> wild type recorded at 465 nm. Peak 1, R[E,M]BChl <i>c</i>; peak 2, R[E,E]BChl <i>c</i>; peak 3, S[E,E]BChl <i>c</i>; peak 4, R[P,E]BChl <i>c</i>; peak 5, S[P,E]BChl <i>c</i>; peak 6, R[I,E]BChl <i>c</i>; peak 7, S[I,E]BChl <i>c</i>; peak 8, R[E,E]BChl <i>e</i>; peak 9, S[E,E]BChl <i>e</i>; peak 10, R[P,E]BChl <i>e</i>; peak 11, S[P,E]BChl <i>e</i>; peak 12, R[I,E]BChl <i>e</i>; peak 13, S[I,E]BChl <i>e</i>. Peaks at the asterisk in panel (B) indicate impurities produced during handling of the sample.</p